CA2095540A1 - Expression of malaria polypeptides - Google Patents

Abstract

The present invention is directed to recombinant expression of malaria polypeptides. Nucleic acid constructs having a domain encoding a malaria polypeptide, preferably SERA or an epitope-containing fragment thereof, downstream from a domain encoding a ubiquitin polypeptide are presented. Additionally, constructs with a third domain encoding an immunomodulator polypeptide, preferably human .gamma.-interferon, and positioned between the first and second domains encoding, respectively, ubiquitin and a malaria polypeptide are also included. The malaria polypeptides of the present invention have utility as vaccine candidates and in immunodiagnostic applications.

Description

w092/08795 1 P~T/U~90/0~61 ~9a54a EXPRESSION OF MALARIA POLYPEPTIDES

BACXGROUND OF THE INVENTION

1. Field of the Disclosure This invention relates to the recombinant production of malaria polypeptides and to the use of such polypeptides as vaccine candidates and in immunodiagnostic applications.

2. Brief Description of Related Art Malaria is a debilitating disease caused by sporozoan protozoa of the genus Plasmodium. At least four species of plasmodia may infect humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae, and the disease is transmitted through the bloodsucking bite of Anopheles mosquitoes.
The life cycle of malaria parasites can be summarized as follows. There are two broad phases: an exogenous phase in mosquitoes consisting of sexual or sporogonic development in which infective sporozoites are produced (through the development of a zygote, ookinete, and oocyst, wherein the latter bursts to release sporozoites), and an endogenous phase in humans resulting from injection of the sporozoites through the saliva of the mosquito and consisting of asexual or schizogonic development in which the end result is the ; differentiation of male and female microgametocytes, , .
~ which are then taken up and ingested by mosquitoes. Once .. . . . .
ingested by the mosquito, fertilization of a female ` ` microgametocyte ~y a male microgametocyte results in a zygote, and the life cycle continues as described.
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The endogenous phase in humans begins with exoerythrocytic multiplication in liver parenchymal cells after which numerous asexual progeny, the merozoites, leave ruptured liver cells, enter the bloodstream, and invade erythrocytes. The erythrocytic or clinical stage (producing trophozoites and schizonts) can coexist with continuing exoerythrocytic multiplication. During the erythrocytic stage, certain merozoites become differentiated into male or female gametocytes. For a general review of the medical parasitology of malaria, see Jawetz, et al., Review of Medical Microbioloqv, Appleton & Lange, Norwalk, CT (1987).
Morbidity and mortality caused by malaria render it a serious problem, and the development of resistance to prophylactic and therapeutic drugs by malaria parasites, and to insecticides by their mosquito vectors, has reaffirmed the need for an effective vaccine against Plasmodium. Initially, it was thought that immune responses targeted to the sporozoite stage, in particular to a circumsporozoite (CS) protein, would provide ;
protective immunity to both visitors to, and inhabitants of, malarious regions, see Young, et al., Science 228:95a-962 (1987). For examples of such sporozoite antigens, see, e.g., U.S. Patent No. 4,466,917; U~S.
Patent No. 4,769,235; PCT Application No. WO 88/05817 (published 11 August 1988); PCT Application No. WO
87/00533 (published 29 January 1987); PCT Application No. WO 86t05790 (published 9 October 1986); PCT
Application No. 84/02922 (published 2 August 1984).
To date, however, vaccine trials in humans with recombinant circumsporozoite antigens have been only modestly successful, see Ballou, et al., Lancet i:l276-1281 (1987); Herrington, et al., Nature 328:257-259 (1987); Gordon, et al., Am. J. Trop. Med. Hyg. 42:527-531 -W092/0X79~ PCT/US9~/~\~61 ,, s .;
` "2~g5~0 (1990); suggesting that alternative approaches ~ay be required for effective malaria vaccination. In addition, strategies for immune evasion adopted by the malaria parasite suggest that multiple immunogens, encompassing material from more than one malarial life cycle stage, will be advantageous in a subunit vaccine cocktail.
Therefore, the efficient production of these vaccine components is a high priority.
In PCT Application No. 90/01549 (published 22 February 1990), an isolated nucleic acid sequence encoding designated SERA (serine~repeat antigen) of Plasmodium falciparum is given.
Perrin, et al., J. Ex. Med. 160:441-451 (1984), report that immunization with a whole P140 protein, purified from cultured schizonts of Plasmodium falciparum SGE2 strain from Zaire, partially protected Saimiri monkeys from a blood stage infection of malaria.

SUMMARY OF THE INVENTION
The present invention is directed toward efficient recombinant expression of malaria polypeptides and includes nucleic acid constructs for such polypeptide expression. These constructs are comprised of a first domain encoding a ubiquitin peptide and a second domain encoding a malaria polypeptide or an epitope-containing fragment thereof, wherein the second domain is located downstream from the first domain. The preferred malaria polypeptide is SERA (serine repeat antigen), more preferably SERA fragments, most preferably, SERA 1 and SERA N, from Plasmodium falci~arum. Additionally, the present invention includes nucleic acid constructs further comprising a third domain encoding an immunomodulator polypeptide, preferably y-interferon, wherein the third domain is positioned downstream from ~.

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w092/0879~ _4_ PCT/US90/0~61 209~0 .
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the first dom~in and, preferably, upstream from the second domai~.
These constructs can be used to transform a variety of host cells, including but not limited to yeast, and this invention encompasses such host cells transformed by the above-described constructs.
Furthermore, processes for producing malaria polypeptides, including but not limited to SERA, using the constructs of this invention are described below.
Fusion polypeptides comprising, for example, an immunomodulator polypeptide and a malaria polypeptide are also included in the present invention.
Malaria polypeptides expressed by the host cells transformed with the constructs described herein have utility in the production of antiserum, particularly in primates, and as vaccine candidates. Immunogenic compositions produced by the processes of the present invention are disclosed, as well as methods for malaria treatment in individuals, and these compositions additionally may include more than one malaria polypeptide and/or an adjuvant, as well as an optional pharmaceutically acceptable carrier.
Furthermore, malaria polypeptides expressed by host cells transformed with the present constructs, as well as antiserum raised against such polypeptides, can be used in immunodiagnostic applications, and such applications are included in the scope of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS - -Fig. 1 refers to representative expression plasmids for the production of SERA polypeptides wherein (a) is pBS24Ub-SERA 1, and (b) is pBS24Ub-yIFN-SERA N.

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DETAILED DESCRIPTION OF THE INVENTION
AND SPECIFIC EMBODIMENTS
1. Definitions The present invention is directed toward nucleic acid constructs comprising a first domain encoding ubiquitin and a second d~main encoding a malaria polypeptide or epitope-containing fragment thereof. Such constructs can be used to transform host cells for the expression of malaria polypeptides. The produced polypeptides have utility as vaccine candidates.
As used herein, "construct" refers primarily to expression vectors, wherein a "vector" identifies a replicon into which another polynucleotide segment is attached, so as to bring about the replication and/or expression of the attached segment. A "replicon" refers to any genetic element, e.g., a plasmid; a virus, including but not limited to, bacteriophage, baculovirus, vaccinia; a chromosome; etc. that behaves as an autonomous unit of polynucleotide replication within a cell.
In the present invention, the attached polynucleotide seqment, is also referred to as a "domain." A first domain or segment is "heterologous"
with respect to an associated second domain or segment when such association (e.g., ligation, occurrence in the same host cell, etc.) does not occur in nature. The constructs of the present invention comprise a first and second domain and, in a preferred embodiment, third domain is present; these domains are specified below.
Additionally, other translational and transcriptional control sequences may be present.
"Control sequence" refers to polynucleotide se~uences that are necessary to effect the expression of coding sequences to which they are ligated. The nature of such ~ .

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control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and terminators; in eukaryotes, generally, such control sequences include promoters, terminators, and, in some instances, enhancers. "Control sequences" is intended to include, at a minimum, all components whose presence is necessary for expression and may also include additional components whose presence is advantageous, for example leader sequences.
The first and second domains as well as any control sequences are operably linked in the present constructs. By "operably linked," it is meant a juxtaposition wherein the components are in a relationship permitting them to function in their intended manner. A control sequence "operably connected"
to a coding sequence, for example, is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences, i.e., through the binding of host cell RNA polymerase to the promoter of the control sequences.
The present constructs can be used to transform -host cells. As used herein, "host cells," "host organisms," "cells," "cell lines," "cell cultures," and other such terms denoting prokaryotic microorganisms or eukaryotic cell lines cultured as unicellular entities are used interchangeably to refer to cells that can be, or have been, used as recipients for recombinant nucleic acid constructs or other transfer nucleic acids and include the progeny of the original cell that has been transformed. Such progeny may not be necessarily identical in morphology or in genomic or total DNA
complement as the original parent; however, progeny sufficiently similar to be characterized by the same 'j' .', ,,,,, . . ' ,, "' .'. ' ' ., . ., ', ,' ' ,'.' . '. ' ' . ' . " ~, ' '' ' ' .:: :'.-' '' ' . ' " ' ' , . . ' ' ' ., . ' , ', . '" ' ~ , :' ~ -, ' '' ' '' . ' .: . ' W092/08795 7 pcT/us9n/o~61 2~5,~

relevant property, such as the presence of a nucleotide sequence encoding a malaria polypeptide, are included.
By "transformation," it is meant the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for insertion, for example, direct uptake, transduction, or f-mating. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid; or, alternatively, may be integrated into the host genome.
A "peptide," "polypeptide," or "protein", used interchangeably herein, means of poymer of amino acids linked through peptide bonds.
As used herein, "malaria polypeptides" refer to any peptides, polypeptides, or proteins encoded in the genome of a Plasmodium species. "Immunogenic" refers to the ability of a polypeptide to cause a humoral and/or cellular immune response, whether alone or when linked to a carrier, in the presence or absence of an adjuvant.
"Neutralization" refers to an immune response that blocks the infectivity, either partially or fully, of an infectious agent.
"Epitope" refers to an antigenic determinant of a peptide, polypeptide, or protein; an epitope can comprise 3 or more amino acids in a spatial conformation unique to the epitope. Generally, an epitope consists of at least 5 such amino acids and, more usually, consists of at least 8-10 such amino acids. Methods of determining spatial conformation of amino acids are known in the art and include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. Antibodies thatrecognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.

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"Treatment," as used herein, refers to prophylaxis and/or therapy (i.e., the modulation of any malaria symptoms). An "individual" indicates an animal that is susceptible to infection by Plasmodium and includes, but is not limited to, primates, including humans. A
"vaccine" is an immunogenic or otherwise capable of eliciting protection against malaria, whether partial or complete, composition useful for treatment of an individual.
All patents, patent applications, and publications mentioned herein, both above and below, are incorporated by reference into this application. Unless otherwise designated, recombinant techniques used are those known to one of ordinary skill in the art. See, e.g., --Maniatis, et al., Molecular Cloninq: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1982); Sambrook, et al., Molecular Clonina: A Laboratory Manual (2d ed. vols. 1-3), Coid Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

2. Modes for Carryina out the Invention The nucleic acid constructs of the present invention, preferably DNA constructs, comprise a first domain encoding for ubiquitin, which is upstream to the second domain encoding a malaria polypeptide or epitope-containing fraament thereof.
Ubiquitin (Ub) is a highly conserved 76 residue protein found in sukaryotes. See, e.g., Rechsteiner ` 30 ~ (ed.), Ubiauitin, Plenum Press, New York (1988);
Varshavsky, et al.,~pp. 109-143 in Yeast Genetic Enaineerina, edited by Barr, Brake, and Valenzuela, Butterworths, New York ~1989); PCT Application No. WO
88~02406 (published 7 April 1988); Finley, et al., Cell -~. .
.
, W092/08795 l~CT/US90/0~61 _g_ 2~.S~O

37:43-55 (1984). Fusion of genes encoding for example human and bovine growth hormones, y-interferon, ~-proteinase inhibitor, acidic fibroblast growth factor, interleukin-2, metallothionein, ~-subunit of human nerve growth factor to ubiquitin sequences has been suggested.
See Sabin, et al., Bio/Technoloqy 7:705-709 (1989), for a review; see also Butt, et al., Proc. Natl. Acad. Sci.
86:2540-2544 (1989); Butt, et al., J. Biol. Chem.
263:16364-16371 (1988); Ecker, J. Biol. Chem. 264:7715-10 7719 (1989)-The present invention is directed to the novel fusion of genes encoding for malaria polypeptides to a ubiquitin sequence and, in a preferred embodiment, to ubiquitin and a third domain encoding an immunomodulator 15 polypeptide, the latter including but not limited to human y-interferon sequences, wherein this third domain is operably linked downstream from the ubiquitin domain and either upstream or downstream from the malaria polypeptide domain, preferably upstream.
The preferred first domain ubiquitin and third domain y-interferon combination described can be linked to polypeptide epitopes of infectious agents other than Plasmodium, in a manner similar to what is disclosed herein.
In one preferred embodiment, the first domain encoding ubiquitin is operably linked to, and under the control of, a regulated, high-level promoter. An example of such a promoter for yeast hosts is a glucose-regulatable, hybrid promoter, such as one comprised of a ~ -30 yeast alcohol dehydrogenase 2 (ADH2) promoter and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. This promoter, ADH2/GAPDH, is described in Barr, et al., J. Exp. Med. 165:1160-1171 (1987).

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W092/08795 _lo_ PCT/US90/0~61 209~P

The second domain of the present constructs encodes a malaria polypeptide or an epitope-containing fragment thereof, as defined above. Examples of malaria polypeptides include, but are not limited to, those associated with sexual phase zygotes and ookinetes, such as surface antigens, including pfs25 of Plasmodium falciparum; those associated with sporozoites, such as circumsporozoite (CS) antigens from various species, e.g., Plasmodium falci~arum and Plasmodium vivax; those associated with asexual phase merozoites, such as a major merozoite surface protein of Plasmodium falciparum designated gpl95 as discussed in Siddiqui, et al., Proc.
Natl. Acad. Sci. USA 84:3014-3018 (1987), Pv200 as mentioned in del Portillo, Exp. Parasitoloay 67:346-353 (1988), and secreted Plasmodium falci~arum SERA (serine-repeat antigen) as described below; etc. For a survey of recent developments in this area, see Program Booklet from The 38th Annual Meeting of the American Society of Tropical Medicine and Hygiene (ASTMH), Honolulu, Hawaii, December 10-14, 1989. See also U.S. Patent No.

4,826,957.
In a preferred embodiment of the present invention, the second domain encodes SERA or an epitope-containing fragment thereof. SERA is an acronym for serine-repeat antigen and has been previously described (and called pl26, pll3, Pfl40, or SERP 1), see review of SERA references below. The DNA sequence for SERA can be found in PC~ Application No. W0 90/01549 (published 22 February l990), Figure 2. SERA is known to contain 989 amino acids, including a putative signal peptide sequence, and it has a molecular weight of 111, 000 Typically, a hydrophobic block of serine residues (polyS) is present. Another notable feature of SERA is a typical hydrophobic signal peptide at the amino (N) terminus.

, - . : :,,, , - ., . . -Wog~/08795 -11- PCT/US90/0~61 2095~0 At least 1.5% of mRNA in trophozoites and schizonts is SERA mRNA. SERA accumulates in the parasitophorous vacuole of erythrocytic Plasmodium falciparum trophozoites and schizonts and is released into culture medium at, or near, the time of merozoite release; at such time, the protein undergoes processing into several, smaller polypeptides. Although SERA is found primarily in parasitophorous vacuoles, it has also been found associated with intraerythrocytic, but not free, merozoites in culture, see, e.g., Chulay, J.
Immunol. 139:2768-2774 (1988); Delplace, Mol. Biochem.
Parasitol. 23:193-201 (1985). The fragility of free merozoites in culture may explain the inability to detect the association in them. SERA is available as a target for several parasite neutralizing murine monoclonal antibodies, such as 43E5, see Banyal, et al., Am. J.
Trop. Med. Hyq. 34:1055-1064. It is postulated that SERA
may have a proteolytic function, see, e.g., Higgins, et al., Nature 340:604 (1989); Eakin, et al., Nature 342:132 (1989); Mottram, et al., Nature 342:132 (1989).
For a review of SERA, see, e.g., PCT Application No. WO 90/01549 (published 22 February 1990); Bzik, et al., Mol. Biochem. Parasitol. 30:279-288 (1988); Li, et al., Mol. 8iochem. Parasitol. 33:13-26 (1989); Delplace, et al., Mol. Biochem. Parasitol. 23:193-201 (1987);
Weber, et al., pp. 379-388 in Molecular Strate~ies of Parasitic Invasion, Alan R. Liss, New York (1987); Knapp, et al., Mo. Biochem. Parasitol. 32:73-84 (1989); Perrin, et al., J. Exp. Med. 160:441-451 (1984); Horii, et al., Nol. Biochem. ~arasitol. 30:9-18 (1988). A cDNA clone of SERA has~been entered in Genebank, see PCT Application No. 90/01549 (published 22 February 1990) and an isolated nucleic acid sequence encoding SERA is given therein.

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w092/ox795 12 PCT/I~S90/06461 ~20'95~a It is to be understood that, although SERA is specified in the literature to be from Plasmodium falciparum, the present invention also includes polypeptides homologous to SERA from other species of Plasmodium. The genes for such homologous polypeptides can be identified by low stringency nucleic acid hybridization using the SERA gene or fragments thereof from Plasmodium falciparum.
It is preferred that epitope-containing fragments of SERA be used in the present constructs and, more preferably, that the SERA epitope-containing fragments are what is designated herein as SERiA 1, corresponding to amino acids 24(Gly)-285 (Asp) of the natural Plasmodium falciparum SERA precursor protein, with the putative signal peptide removed, and having a molecular weight of 26,976; SERA N, a larger peptide including SERA 1 .
corresponding to amino acids 24(Gly)-506(Pro) of the precursor protein and having a molecular weight of 52,520; and any epitope-containing fragments of SERA 1 : -and SERA N .
In addition to a first domain encoding ubiquitin and a second, downstream domain encoding a malaria polypeptide or an epitope-containing fragment thereof, a third domain optionally may be present. This domain encodes an immunomodulator polypeptide, which is defined to include any protein, polypeptide, or peptide that has an effect on humoral or cellular immune response. As .
such, immunomodulator polypeptides include, but are not limited to interleukins. IL1-11, tumor necrosis factor (TNF), colony stimulating factors (CSF), and interferons (IFN). Of particular interest in the present constructs is human gamma-interferon (y-IFN), particularly in combination with SERA N (i.e., ~-IFN-SERA N). The third domain is operably linked downstream from the first .
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r W092/~8795 PCT/IIS90/0~61 .-` 2~5~3 domain and either upstream or downstream from the second domain, preferably upstream.
Playfair, et al., Clin. Exp. Immunol. 67:5-lo (1987) report, using a rodent malaria system, that recombinant y-interferon is a potent adjuvant for a malaria vaccine in mice. Sturchler, et al., Vaccine 7:457-461 (1989), report a human vaccine having a synthesized epitope of the circumsporozoite (CS) protein of Plasmodium falciparum conjugated to tetanus toxoid as a carrier protein and employing ~- or y-interferon as an adjuvant.
Representative constructs are given in Fig. 1, wherein (a) is plasmid pBS24Ub-SERA 1 and (b) is pBS24Ub-yIFN-SERA N. The construction of pBS24Ub, having a ADH2/GAPDH hybrid promoter, is described in Sa~in, et -al., Bio/Technoloay 7:705-709 (1989).
In general, the malaria polypeptides of this invention may be expressed in vitro, or in vivo in either prokaryotic or eukaryotic systems. Alternatively, the polypeptides can be produced by chemical synthesis. Host cells transformed with the above-described constructs are included in the present invention as are processes for expressing malaria polypeptides and fusion polypeptides.
Prokaryotes are most frequently represented by various strains of Escherichia coli. However, other microbial strains may also be used, such as bacilli (for example Bacillus subtilis), various species of Pseudomonas, and other bacterial strains. In such prokaryotic systems, plasmid vectors that contain replication sites and control sequences derived from a species compatible with the host can be used. For example, E. coli may be typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species by Bolivar, et al., Gene 2:95 (1977). Commonly ' ~ ~
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used prokaryotic control sequences, which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, can include such commonly used promoters as the ~-lactamase (penicillinase) and lactose (lac) promoter systems, see Chang, et al., Nature 198:1056 (1977,) and the tryptophan (trp) promoter system, see Goeddel, et al., Nuc. Acids Res. (1980) 8:4057, and the lambda-derived PL promoter and N-gene ribosome binding site, Shimatake, et al, Nature 292:128 (1981). However, --any available promoter system compatible with prokaryotes can be used.
The expression systems useful in eukaryotic systems of the invention can comprise promoters derived from appropriate eukaryotic genes. A preferred eukaryotic host is yeast, preferably Saccharomyces (e.g., ; Saccharomyces cerevisiae) or Kluyveromvces (e.g., Kluyveromyces lactis). A class of promoters useful in yeast, for example, includes promoters for synthesis of glycolytic enzymes, including those for -3-phosphoglycerate kinase, see Hitzeman, et al., J. Biol.
Chem. 255:2073 (1980). Other promoters include those from the enolase gene, see M.J. Holland, et al., J. Biol.
Chem. 256:1385 (1981) or the Leu2 gene obtained from YEpl3, see J. Broach, et al., Gene 8:121 (1978). As mentioned above, the hybrid promoter, ADH2/GAPDH is most preferred in the present invention.
Suitable mammalian promoters can include the early and late promoters from SV40, see Fiers et al, Nature 273:113 (1978) or other viral promoters, such as those derived from polyoma, adenovirus II, bovine papilloma virus, or avian sarcoma viruses. In the event plant cells are used as an expression system, the nopaline ~; synthesis promoter is appropriate, see Depicker, et al., .

5 ~ 5 PCr/ ~!S90/0646 1 : ~ 2~a~

J. Mol. Appl. Gen. 1:561 (1982). Expression in insect cell culture conveniently may be achieved using a baculovirus vector, see, e.g., Luckow, et al., Bio/Technoloay 6:47-55 (1988).
Depending on the host cell used, transformation can be done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described by Cohen, Proc. Natl. Acad. Sci. USA (1972) 69:2110 (1972), or the RbCl method described in Maniatis, et al., Molecular Clonina: A LaboratorY Manual Cold Spring Harbor Laboratory Press (1982) at page 254, can be used for prokaryotes or other cells that contain substantial cell wall barriers. Infection with AqrobaGterium tumefaciens, see Shaw, et al., Gene 23:315 (1983) can ~e used for certain plant cells. For mammalian cells without cell walls, the calcium phosphate precipitation method of Graham, et al., Virology 52:546 (1978) is preferred. Transformations into yeast may be carried out according to the method of Van Solingen, et -al., J. Bacter. 130:946 (1977) and ~siao, et al., Proc.
Natl. Acad. Sci USA 76:3829 (1979). Alternatively, one may use a liposomal transfection system. For example, one may use a synthetic lipid such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, commercially available under the name Lipofectin (8RL, Gaithersburg, MD), as described Felgner, et al., Proc. Natl. Acad. Sci. USA 84:7413 (1987).
Construction of suitable vectors containing the present first, second, and optional third domains and control sequences employs standard ligation and restriction techniques, which are well understood in the art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides can be cleaved, tailored, and religated in the form desired.

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Site-speoific DNA cleavage can be performed by treatment with a suitable restriction enzyme (or enzymes) under conditions generally understood in the art, generally following the manufacturer's directions. See, e.g., New England Biolabs Product Catalog. In general, about 1 ~g of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 ~l of buffer solution;
typically, an excess of restriction enzyme is used to ensure complete digestion of the DNA substrate.
Incubation times of about 1 hr to 2 hr at about 37C are worXable, although variations can be tolerated. After each incubation, protein is removed by extraction with phenol/chloroform, and may be followed by diethyl ether extraction, and the nucleic acid recovered from aqueous fractions by ethanol precipitation followed by separation over a Sephadex G-50 spin column. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Meth. Enzvmol. 65:499-560 (1980).
Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA
polymerase I (Klenow) in the presence of the four deoxyribonucleotide triphosphates (dNTPs) using incubation times of about 15 to 25 min at 20 to 25C in 50 mM Tris, pH 7.6, 50 mM NaCl, 6 mM MgCl2, 6 mM DTT and 5-10 ~M dNTPs. The Klenow fragment fills in at 5' sticky ends but chews back protruding 3' single strands, even though the four dNTPs are present. If desired, selective repair can be performed by supplying only 1-3 of the dNTPs, within the limitations dictated by the nature of the sticky ends. After treatment with Klenow fragment, the mixture can be extracted with phenol/chloroform and ethanol precipitated followed by running over a Sephadex ~ .
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G-50 spin column. Treatment under appropriate conditions with Sl nuclease results in hydrolysis of any single-stranded portion.
Synthetic oligonucleotides can be synthesized using the phosphoramidite method on automated oligonucleotide synthesizers, such as the Applied Biosystems 380A DNA synthesizers. Kinasing of single strands prior to annealing or for labeling can be achieved using an excess, e.g., approximately 10 units of polynucleotide kinase to 0.1 nmole substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgC12, 5 mM
dithiothreitol, 1-2 mM ATP, 1.7 pmoles P-ATP (2.9 mCi/mmole), 0.1 mM spermidine, and 0.1 mM EDTA.
Ligations can be performed in 15-30 ~1 volumes under the following standard conditions and temperatures: -20 mM Tris-HCl, pH 7.5, 10 mM MgC12, 10 mM DTT, 33 ~g/ml BSA, 10 mM-50 mM NaCl, and either 40 ~M ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0C (for "sticky end"
ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA
ligase at 14C (for "blunt end" ligation).
Intermolecular "sticky end" ligations can be performed at 33-100 ~g/ml total DNA concentrations (5-100 nM total end concentration). Intermolecular blunt end ligations (usually employing a 10-30 fold molar excess of linkers) are performed at 1 ~M total ends concentration.
In vector construction employing "vector fragments," the vector fragment can be treated with bacterial alkaline phosphatase (BAP) in order to remove the 5' phosphate and prevent religation of the vector.
BAP digestions are conducted at pH 8 in approximately 150 mM ~ris, in the presence-of Na and Mg2 using about 1 unit of BAP per ~g of vector at 60C for about 1 hr. In ~ order to recover the nucleic acid fragments, the - preparation can be extracted with phenol/chloroform and , ~ , . ., :
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w092/08795 18 Pcr/u~sn/o6461 2~9~

ethanol precipitated and desalted by application to a Sephadex G-50 spin column. Alternatively, religation can be prevented in vectors which have been double digested by additional restriction enzyme digestion of the 5 unwanted fragments.
For portions of vectors derived from cDNA or genomic DNA that require sequence modifications, site specific primer directed mutagenesis may be used. This is conducted using a synthetic oligonucleotide primer complementary to a single-stranded phage DNA to be mutagenized except for limited mismatching, representing the desired mutation. Briefly, the synthetic oligo-nucleotide can be used as a primer to direct synthesis of a strand complementary to the phage, and the resulting double-stranded DNA is transformed into a phage-supporting host bacterium. Cultures of the trans-formed bacteria are plated in top agar, permitting plaque formation from single cells which harbor the phage.
Theoretically, 50% of the new plaques will contain the phage having the mutated form as a single strand; 50%
will have the original sequence. The resulting plaques are hybridized with kinased synthetic primer under allele-specific conditions. In general, one may vary the temperature, ionic strength, and concentration of chaotropic agent(s) in the hybridization solution to obtain conditions under which substantially no probes will hybridize in the absence of an "exact match." For hybridization of probes to bound DNA, the empirical formula for calculating optimum temperature under standard conditions (0.9 M NaCl) is T(C) = 4(NG + Nc) +
2(NA + NT) - 5C, where NG, Nc, NA, and NT are the numbers of G, C, A, and T bases in the probe, see Neinkoth, et al., Anal. Biochem. 138:267-84 (1984).
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W0~2/0879; _19_ PCTtU~90/n6461 2~9~

Plaques that hybridize with the probe are then picked, cultured, and the DNA recovered.
Correct ligations for plasmid construction may be confirmed by first transforming E. coli strain MM294 obtained from E. coli Genetic Stock Center, CGSC #6135, or other suitable host with the ligation mixture.
Successful transformants can be selected by ampicillin, tetracycline, or other antibiotic resistance or using other markers depending on the plasmid construction, as is understood in the art. Plasmids from the transformants can be then prepared according to the method of Clewell, et al., Proc. Natl. Acad. Sci. USA
62:1159 (1969), optionally following chloramphenicol amp-lification, see Clewell, J. Bacteriol. 110:667 (1972).
The isolated DNA can be analyzed by restriction and/or sequenced by the dideoxy method of Sanger, et al., Proc.
Natl. Acad. Sci. USA 74:5463 (1977), as further described by Messing, et al., Nuc. Acids Res. 9:309 (1981), or by the method of Maxam, et al., Meth. EnzYmol. 65:499 (1980).
The present invention also includes fusion polypeptides and processes for expressing such malaria polypeptides and malaria fusion polypeptides. Fusion polypeptides comprise an immunomodulator polypeptide fused to a malaria polypeptide. With a yeast host, in vivo cleavage by endogenous yeast ubiquitin hydrolase occurs, and the mature polypeptide can be subsequently isolated. In other hosts, ubiquitin hydrolase (UbH), which is commercially available, can be used to cleave off ubiquitin, preferably during polypeptide purification; such techniques are ~nown to one of skill in the art, see, e.g., Butt, Proc. Natl. Acad. Sci. USA
86:2540-2544 (1989); Miller, at al., Bio/Technoloa~
7:698-704 (1989).

W092/0~795 PCT/~IS90/0~61 Z O 9 ~

In preferred embodiments, the fusion polypeptides are comprised of SERA N fused to y-IFN, (designated herein as y-IFN-SERA N), resulting in a fusion protein with a molecular weight of 69,224; SERA 1 fused to y-IFN
(y-IFN-SERA 1) resulting in a fusion protein with a molecular weight of 43,680. As yeast ubiquitin is cleaved, the molecular weight of ubiquitin is not included in these molecular weight calculations.
Processes for increasing the level of expression of a recombinant malaria polypeptide or an epitope-containing fragment thereof in a host cell comprise the steps of: (a) constructing a vector consisting of a first domain encoding ubiquitin and a second domain encoding an malaria polypeptide or epitope-containing fragment thereof, wherein the second domain is located downstream from the first domain; (b) transforming the host cell with the constructed vector of (a); (c) culturing the transformed host cell under conditions capable of inducing expression of the ubiquitin-malaria domains ~-whereby the resulting in vivo product is a recombinant malaria polypeptide; and (d) recovering the recombinant malaria polypeptide from the transformed host culture.
Conditions capable of inducing expression in a host cell are known in the art. Preferably, the malaria polypeptide is a SERA epitope-containing fragment, more preferably SERA 1, and the host cell is yeast. When the host cell is yeast, a preferred set of conditions involves transformation of yeast cells by the spheroplast method, see, e.g., Hinnen, et al., Proc. Natl. Acad. Sci.
USA 75:1929-1933 (2978), and propagation under conditions of leucine selection prior to liquid culture in YEP
media.
Furthermore, the present invention includes ~; processes for increasing the level of expression of a -W092/0879S pcr/l~9o/o~6 2~S5~ '3 recombinant malaria polypeptide or an epitope-containing fragment thereof, in a host cell comprising: (a) constructing a vector consisting of a first domain encoding ubiquitin, a second domain downstream ~o the first domain encoding the malaria polypeptide or epitope-containing fragment thereof, and a third domain located between the first and second domains encoding an immunomodulator; (b) transforming the host cell with the constructed vector of (a); (c) culturing the transformed host cell under conditions capable of inducing expression of the ubiquitin-immunomodulator-malaria domains, whereby the resulting in vivo product is a recombinant immunomodulator-malaria fusion polypeptide; and (d) recovering the recombinant fusion polypeptide from the transformed yeast culture. In a preferred embodiment, the malaria polypeptide is a SERA fragment, more preferably SERA N; the immunomodulator is y-interferon;
and the host cell is yeast.
Protein purification methods are known in the art.
Characteristics of a polypeptide, such as solubility, charge, hydrophobicity, and intermolecular bonding, influence the choice of purification method as the conformation of a polypeptide provides specific binding properties between the polypeptide and other molecules.
Initially, polypeptides can be separated from low molecular weight substances present in a cell by the ~ -process of dialysis or solvent extraction. Polypeptides then can be sorted on the basis of size through, for example, gel filtration. Polypeptides also can be separated through electrophoresis on the basis of the charge. Ion-exchange chromatography, based upon density and charge of molecules, can also be used.
In the present case, a preferred purification method is as follows. The malaria fusion polypeptide, W092/0879~ -22- PCT/~'SgO/0~61 209~5~0 preferably y-IFN-SERA N, is first subjected to a solvent extraction utilizing urea and SDS (sodium dodecyl sulfate), followed by an ion-exchange step. Lastly, separation and purification is accomplished through gel filtration. For a malaria polypeptide, preferably SERA
1, the preferred process is as described above, except that the ion-exchange step is eliminated.
Malaria polypeptides and fusion immunomodulator-malaria polypeptides, preferably ~-IFN SERA polypeptides, have utility in immunogenic compositions, vaccines, and in immunodiagnostic applications. The recombinant polypeptide products of this invention can be expressed in a quantity sufficient for efficient vaccine manufacture and immunodiagnostic assays, and also do not contain impurities that may be found in polypeptides collected from in vitro parasite cultures.
Immunogenic compositions can be prepared according to methods known in the art. The present compositions comprise an immunogenic amount of a polypeptide, i.e., a malaria polypeptide or a fusion immunomodulator-malaria polypeptide or a cocktail of more than one malaria polypeptide, usually admixed with a pharmaceutically acceptable carrier, preferably further comprising an adjuvant. If a "cocktail" is desired, a combination of malaria polypeptides, such as, for example, SERA N plus circumsporozoite antigens, can be mixed together for heightened efficacy.
Pharmaceutically acceptable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers; and ~- .

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inactive virus particles. Such carriers are well known to those of ordinary skill in the art.
Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to:
aluminum hydroxide (alum), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) as found in U.S. Patent No.
4,606,918, N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine (MTP-PE) and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. Alum and a muramyl tripeptide formulation MF59, as described in the Examples, are preferred.
Additionally, adjuvants such as Stimulon (Cambridge Bioscience, Worcester, MA) may be used. Furthermore, Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA) may be used for non-human applications, see Examples section below.
Furthermore, the immunogenic compositions typically will contain pharmaceutically acceptable vehicles, such as water, saline, glycerol, ethanol, etc.
Additionally, auxiliary substances, such as wetting or emulsifying agents, Ph buffering substances, and the like, may be present in such vehicles.
Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect.

,- ,' W092/08795 PC~/US90~0~6 2 0 9~ Q

Immunogenic compositions used as vaccines comprise an immunologically effective amount of the malaria polypeptide or fusion polypeptide, as well as any other of the above-mentioned components, as needed. By "immunologically effective amount", it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment, as defined above. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g., nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, the species of infecting Plasmodium, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
The immunogenic compositions are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include oral formulations and suppositories. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.
The present malaria polypeptides and fusion polypeptides also have utility in immunodiagnostic applications. In general, (a) a biological sample suspected of containing malaria antibodies can be incubated with a malaria polypeptide or fusion polypeptide of the present invention under conditions that allow the formation of an antibody-antigen complex, ,~
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W092/0~795 PC~/US9~/0646 2-~ 9~
and (b) then the antibody-antigen complex can be detected by methods known to a skilled practitioner. Design and protocol of such immunoassays is subject to a great deal of variation and many methods are known in the art.
Protocols may be based, for example, upon competition, direct reaction, or sandwich type assays. The malaria polypeptide or fusion protein may be fixed on a solid support, for example. Labeled antibodies may be used for detection, as in enzyme-linked immunosorbent assays (ELISA). Additionally, Western blots can also be used in immunodiagnostic applications.
In addition, antisera produced against the above malaria polypeptides and fusion polypeptides will have obvious utility as immunological reagents for, e.g., purification, titration of protein, diagnosis, taxonomic identification, etc.

3. Examples The examples presented below are provided as a further guide to the practitioner of ordinary skill in the art and are not to be construed as limiting the invention in any way.
Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific materials and components -described herein. Such equivalents are intended to be encompassed in the scope of the appended claims.
In these Examples, it has been shown that certain defined regions of the SERA protein can be efficiently expressed in yeast, Saccharomyces cerevisiae. and polyclonal antisera generated in mice against these purified recombinant domains of the SERA protein were ! capable of neutralizing parasites in an in vitro assay.
~ Furthermore, the vaccination of Aotus monkeys with SERA 1 . .
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W092/08795 -26- PC~/lSgo/~61 2095r~
i and y-IFN-SERA N showed that these recombinant polypeptides stimulated a strong protective immunity when administered with Complete and Incomplete Freunds Adjuvants (CFA and IFA). The produced immunity inhibited parasite growth in monkeys challenged by intra~enous inoculation with blood stage Plasmodium falciparum.

3.a. Clonina and Construction of Expression Vectors Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs a~d Boehringer Mannheim. AmpliTaq kits were purchased from Perkin-Elmer/Cetus. Oligomers were synthesized by the phosphoramidite method on Applied Biosystems 380A DNA
synthesizers. The plasmid pTSE/SERA, described in Li, et al., Mol. Biochem. Parasitol. 33:13-26 (1989); Bzik, et al., Mol. Biochem. Parasitol. 30:279-288 (1988), was used for template DNA in polymerase chain (PCR) reactions.
Plasmodium falciparum Honduras-1 strain SERA CDNA
was configured for insertion into yeast expression vectors by the PCR method of Schaarf, et al., Science 233:1076-1078 (1986). For direct expression using the hybrid ADH2/GAPDH promoter, 5'-primers were designed with an in-frame methionine initiation codon and an Ncol restriction site at the 5' end of each gene. For example, the 5'-primer for SERA 1 and SERA N was the following: 5'-ATA.AAA.TCC.ATG.GGA.GAA.AGT.CAA.ACA.
GGT.AAT.-3'. A Sall site, following a stop codon at the 3' end of the gene, was included in each 3'-primer to enable the cloning of the amplified SERA DNA fragment as, for example: 3'-CCT.TTA.TTG.TTT.CAA.CTA.ATC.AGC.TGG.
CCA.ATT-5', the 3'-primer for SERA 1.
The PCR products were cloned into the plasmid pBSlO0, see Barr, et al., J. Exp. Med. 165:1160-1171 (1987). For Ub fusions, a similar PCR strategy was used.

Wos2/08795 Pcr/os9o/()~6 - ~ ~ '3 ~

SstII sites were inserted using PCR primers for the junction of the Ub and SERA sequences; this insertion is described in Sabin, et al., BioTechnologv 7:705-709 (1989). The 3'-primer was downstream of the stop codon within the GAPDH terminator of the pBS100 SERA
constructs. For Uby-IFN fusion vector construction, the above NcoI/SalI fragments were cloned into a Uby-IFN
vector that had been previously modified by in-frame insertion of a NcoI site at the 3' -end of the y-IFN
sequence.
Expression levels varied over a wide range, with amino-terminal domains being produced at the highest levels. Based on the reactivity of murine monoclonal antibodies 43E5 with amino-terminal regions of SERA, together with the observed high levels of expression of these regions, it was determined that SERA 1 and y-IFN- -SERA N would be purified for further immunological ---analysis.
Plasmids for production of SERA 1 and y-IFN-SERA N
are shown in Fig. 1 (a) pBS24Ub-SERA 1 and (b) pBS24Ub-y-IFN-SERA N.

3.b. Recombinant SERA Polvpe~tide Ex~ression Yeast cells of Saccharomvces cerevisiae were transformed with the above-identified plasmids and induced for parasite polypeptide expression as described in Barr, et al., J. Exp. Med. 165:1160-1171 (1987).
Expression was monitored by yeast cell lysis and analysis of both whole cell lysates, and Triton X-100 lysis buffer-soluble and -insoluble fractions. Analysis was performed by SDS-PAGE using Coomassie blue staining and Western blot detection.

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W092/08795 -28- PCT/US90/0~61 2n9~54Q

3.c. Poly~e~tide Purification SERA 1 was purified from transformed yeast cells by glass bead lysis in 50mM Tris/HC1, 62.5mM EDTA, 0.1%
Triton X-100 at pH 8Ø The lysate was centrifuged and the pellet washed once in lysis buffer. The resulting pellet was extracted first with lOOmM Tris/HCl, containing 8mM urea and lmN EDTA at pH 7.0 and then with 2x PBS containing, lmM EDTA and 0.1% SDS. The two supernatants were combined and dialyzed against 2x PBS
containing 0.1% SDS. This material was subjected to gel filtration on Pharmacia S-200 HR. SERA 1 eluted in the void volume, presumably as an aggregate. Appropriate fractions were made lOmM with 2-mercaptoethanol and heated to 80C for 15 min, and then rechromatographed on the same S-200 HR column in 2x PBS containing 0.1% SDS, and lmM 2-mercapthoethanol. Fractions containing SERA 1 were pooled, concentrated and stored in P8S containing 0.1% SDS.
y-IFN-SERA N was purified in a similar manner to SERA 1 with the following modifications. The y-IFN-SERA
N was solubilized in the 8M urea-containing buffer (above) and no SDS extraction was performed. The first gel filtration column was developed in the presence of 2-mercaptoethanol and this was followed by ion exchange chromatography on Pharmacia Fast Flow Q in lOOmM Tris/HCl pH 7.0 containing 8M urea, and lmM EDTA, and elution with a gradient from 0 to O.SM NaCl. The peak fractions containing y-IFN-SERA N were pooled, and rechromatographed by gel filtration in 2x PBS containing ~' 30 0.1% SDS and 2mN 2-mercaptoethanol. The peak fractions were pooled, concentrated and stored at 4C.
Polypeptide purification was monitored by SDS-PAGE
and final concentrations were measured by the Pierce BCA
assay. Amino acid analysis, N-terminal se~uence :: `
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W092/087~ -29- Pcr/us9n/o~6l 2 ~

analysis, and Western blot analysis using the murine monoclonal antibody 43E5 was used to confirm the identity of each polypeptide.
The results showed that both polypeptides gave predominantly single bands by SDS-PAGE analysis. Each purified polypeptide reacted with the monoclonal antibody 43E5 by Western blot analysis. The purity and composition of these two polypeptides were confirmed by amino acid analysis and N-terminal sequencing. For SERA
1, N-terminal amino acid sequence analysis (10 residues) gave the sequence predicted by the SERA DNA sequence and the known cleavage specificity of the yeast ubiquitin hydrolase (UbH). The first amino acid, Gly, was present at greater than 90% yield.
y-IFN-SERA N was determined to be less pure by densitometric scanning of the Coomassie blue stained gel (ca. 79%), and by quantitative amino acid sequence analysis (84-86% purity). The most prominent impurity was reactive with the monoclonal antibody 43E5 by Western blot analysis, and most likely results from carboxyl-terminal degradation. This was further borne out by amino-terminal sequence analysis that showed the first amino acid to be Gln, and was further consistent with the known sequence of human y-IFN over the next 8 residues.

3.d. ~-IFN Activitv Assav Recombinant y-IFN and the y-IFN-SERA N fusion polypeptides were assayed by the microtiter method described previously for the reduction of cytopathogenicity of vesicular stomatitis virus (VSV), see Rubenstein, et al., J. Virol. 376:755-758. Human foreskin fibroblasts (5x104 cells/well) were grown in microtiter plates for 20-24 h with 2 fold serial .
' ' , ' ~ ' W092/0879~ 30 PCI/~JS90/0~61 2~95~i~9 dilutions of the samples to be tested for y-IFN activity.
103 pla~ue forming units of the Indiana strain of VSV
were added per well and the plates incubated for 48 h.
Titers were scored as the reciprocal of the dilution in the well at which 50% of the cell monolayer was protected. Assays were standardized using ~- and y-IFN
obtained from the National Institute of Health (NIH).
y-IFN-SERA N was also assayed for y-IFN activity.
At the assay concentration of 0.5 mg/mL, the activity of y-IFN-SERA N was 18,094 U/mg. SERA 1, used as the control, gave an activity of less than 2,200 U/mg.

3.e. Immunization of Mice Each purified polypeptide was used to immunize mice with either Complete or Incomplete Freunds Adjuvant (CFA or IFA) or a muramyl tripeptide (MTP) adjuvant that has been used in humans. Sera from immunized mice were shown to be capable of in vitro inhibition of invasion of erythrocytes by the Honduras-l strain of Plasmodium falciparum.
Swiss Webster mice were immunized intramuscularly with 3 injections, each separated by 2 weeks. Each injection contained 50 mg of polypeptide in 100 mL.
Groups of four mice received either Complete Freunds Adjuvant (CFA) followed by Incomplete Freunds Adjuvant (CFA/IFA) or a muramyl tripeptide-based adjuvant system, designated NF59. The MF59 adjuvant emulsion contained 5%
squalene, 0.5% Tween 80, 0.5% Span 85 and 400 mg/ml MTP-PE (Ciba-Geigy CG 19835A) in PBS. The emulsion was formulated using a Model llOY microfluidizer (Microfluidics, Newton, MA 02164).
The groups of mice were as follows: 1, SERA
l/CFA/IFA; 2, SERA l/MF59; 3, y-IFN-SERA N/CFA/IFA; 4, y-IFN-SERA N/MF59. Mice were immunized at days 0, 14 and :... : . :- : . - . . . :.

W092~0879~ 31 PCT/US90/0~61 2~9~

28 and their serum antibody titers against SERA
polypeptides were measured by ELISA against the homologous antigen at A, Day 0; B, Day 21; and C, Day 35.
Titers were calculated at 0.2 OD using a V-max microtiter plate reader (Molecular Devices, Palo Alto, Ca.) programmed to read at 650 nm with subtraction of the 490 nm reading.
All titers were less than 100 at prebleed and rose to between 100,000 and 400,000 for the final bleed.
Averaged y-IFN-SERA N titers (386,000 for the CFA/IFA
group and 349,000 for the MTP group) were somewhat higher than the average SERA 1 titers (244,000 for the CFA/IFA
group and 138,000 for the MTP group).
Although the y-IFN-SERA N molecule contains a larger proportion of the full length SERA protein than SERA 1, no large quantitative differences were noted in either antibody titers or neutralization capacity of sera from each group of immunized mice. Although these ~:
parameters were somewhat higher for y-IFN-SERA N, it is unlikely that this is related to the presence of the ~-IFN fusion partner, because human y-IFN is not active in -rodent systems.

3.f. Ervthrocyte Invasion Assav Plasmodium falciparum strain Honduras-1 was maintained in culture according to the method of Trager, et al., Science 193:673-675. Sera from mice immunized with SERA 1 and y-IFN-SERA N were tested for the inhibition of invasion of cultured human erythrocytes by merozoites.
Trophozoite- and schizont-containing erythrocytes were isolated by Plasmagel fractionation, see Banyal, et -~
al., Am. J. Tro~. Med. Hva. 34:1055-1064 (1985), and used at a concentration of 106 infected erythrocytes in the ~

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w092/0879~ -32- PCT/US90/0~61 2 ~ 3 ~ U

presence of 108 normal erythrocytes per ml. Incubations were performed for 72 h, at 37OC, in a final volume of 200 ml. Immune sera were preabsorbed with uninfected human erythrocytes and were assayed at a dilution of 1:20.
Samples were taken at 24, 48 and 72h and Giemsa-stained thin smeared slides were prepared. A
minimum of 5,000 erythrocytes were counted on each slide.
Percentage parasitemia is expressed as the number of lo infected red blood cells divided by the total number of erythrocytes counted X100~. Inhibition of invasion is expressed as the number of infected erythrocytes in the sample culture divided by the number of infected erythrocytes in the corresponding cultures that were incubated with prebleed sera X100~.
Sera were preabsorbed with uninfected human erythrocytes to remove an inhibitory factor that was observed in untreated prebleed sera. Ascites fluid containing the monoclonal antibody 43E5, diluted 1:20, completely abolished the parasitemia, while control cultures, which started at 1.1% parasitemia, increased to 2.6% at 72h. The results showed that control media with no serum addition gave parasitemia levels of 1.1, 1.7, 1.9 and 2.9% at 0, 24, 48 and 72 h respectively. Sera from individual mice or pooled sera were used at 1:20 dilution. At an identical dilution (1:20) of the murine monoclonal antibody ascites fluid 43E5, <0.01%
parasitemia was observed at all time points.
In sum, the results showed that pooled sera, or sera from individual mice injected with SERA 1 or ~-IFN-SERA N could, over the 3 day assay period, significantly reduce or abolish parasitemia in erythrocytes. In the Freunds Adjuvant group, inhibition of parasitemia at 24 h ranged from 50-63~ using sera from SERA 1 immunized ' , , .
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animals, and 60-74% using the sera from y-IFN-SERA N
immunized animals. At the same time point, in the muramyl tripeptide adjuvant group, inhibition of parasitemia ranged from 50-68% using sera from SERA 1 immunized animals, and 51-73% using sera from y-IFN-SERA
N immunized animals. At the 72 h time point only 4 of the 16 groups assayed with individual sera still contained evidence of erythrocyte invasion. Of these four, inhibition of parasitemia ranged from 80-95%.
Similarly, with pooled sera from each group, only in the SERA 1/MF59 group as any parasitemia detected at 72 h.
Here, invasion was inhibited by 98%.
.
3.q. Immunization of Aotus monkeys ; 15 The following example demonstrates that SERA can provide strong immunologic protection in Aotus monkeys against Plasmodium falciparum Honduras-l strain.
Panamanian Aotus lemurinus lemurinus were maintained in the animal facility of Gorgas Memorial Laboratory in Panama City, Panama. A total of 10 animals (adult males and females, weighing from 759 to 970 grams) were divided into three groups of three each that were injected intramuscularly with antigen(s) and/or adjuvant and one group of one monkey that was used as a completely naive control. The monkeys had no previous exposure to Plasmodium falciparum.
SERA 1 and y-IFN-SERA N were used in this experiment. SERA 1 was dissolved in PBS and 0. 05% SDS
whereas y-IFN-SERA N was dissolved in PBS and 0.1% SDS.
Both antigens were dissolved at 800 ~g/ml. The purified antigens, were injected intramuscularly on days 0 and 21 of the experiment. Each dose of antigen was in a final volume of 0.5 ml that contained 200 ~g of the appropriate w092/08795 _34_ PCT/US~0/0~61 2~5~0 antigen(s) in 0.25 ml that was mixed with 0 .25 ml of adjuvant immediately before a vaccination injection.
Monkeys in Groups 1, 2, and 3 received Complete Freunds Adjuvant (CFA) in the primary injection and Incomplete Freunds Adjuvant (IFA) in the booster injection. Monkeys in Group 3 received the antigen carrier solution mixed with the Freunds Adjuvant in each injection. The monkey in Group 4 received neither antigen or adjuvant. Each dose for injection was divided into two 0.25 ml portions and injected intramuscularly into two sites in one thigh of the monkey. All animals were bled 5 days prior to the start of immunization on day 0, 18 days after the primary vaccination injection, and 21 days after the single booster injection, which was just prior to parasite challenge. The development and course of infection was monitored in all monkeys for 100 days after challenge, and the animals were accordingly bled at appropriate times after parasite challenge.
The Plasmodium falciparum Honduras-1 strain used for challenge had been passaged through splenectomized and then nonsplenectomized Aotus monkeys until it grew normally in the latter. ~wenty-two days after the booster injection, each monkey was injected intravenously with 5x104 parasites. Parasitized erythrocytes from an infected monkey with a moderate and increasing parasitemia were used in the challenge to ensure the parasite Yiability- After challenge, the parasitemia was monitored daily by both thick and Earle-Perez films stained with Giemsa. Evaluation of parasitemia was as follows: negative, if ~o parasites were seen after examining a thick blood film for at least five minutes;
<lO parasites per mm3, if parasites could be demonstrated only in a thick blood film; and the number of parasites per mm3 was determined by the Earle-Perez method.

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w092/ox79s -35- PCT/USsO/0~61 2~9~

An ELISA assay was performed. Absorbance was measured at 650 nm. The pre-immunization serum of each monkey was used as the control for each post-immunization serum.
The results were as follows. Two (12504, NM12518) of three monkeys immunized with SERA 1 antigen (Group 1) -developed a peak parasitemia of fewer than 10 parasites/mm3 and the third monkey (12517) developed a peak parasitemia of about 1000/mm3 within the first 20 days after challenge. Two (12512, 12511) of three monkeys immunized with y-IFN-SERA N (Group 2) developed a peak parasitemia of fewer than 10 parasites/mm3 during the first 20 days and subsequently one of these (12512) then developed a brief parasitemia with a peak of 590 parasites/mm3 between days 69 and 73. The third monkey (12519) developed a parasitemia of about 800/mm3 with a --brief peak of 4000/mm3 during the first 20 days.
Monkeys 12517 (Group 1) and 12512 (Group 2) continued to show parasitemias that fluctuated between less than 10 parasite/mm3 and undetectable levels during much of the 100-day period, whereas the other four immunized monkeys from Groups 1 and 2 had, with one brief exception ~NM12518) for 5 days, no detectable parasitemias during the period following the original peak parasitemias.
Two of three monkeys in the control group that received only Freunds Adjuvant (Group 3) developed much higher peak parasitemias than did those monkeys in Groups 1 and 2. The monkey in Group 4 that received neither an antigen nor an adjuvant injection developed a peak parasitemia that was also much higher than that of the monkeys in Groups 1 and 2. All the peak parasitemias in the control groups also occurred within the first 20 days of challenge. After the subsidence of initial i; ' " ~; ' . ' . . ' ' . ' ' . ' , ' ~ .
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w092/0879~ PCT/US90/06461 2a~5~

parasitemias in all controls, no parasites were subsequently seen. Not only were the parasite levels in the two monkeys from Groups 1 and 2 much lower than those in the controls, but the time during which parasitemias in monkeys were greater than 500/mm3 was short compared to the times of higher parasitemias in Group 3 and 4 monkeys. These results suggest that the early parasite loads experienced by the challenged monkeys may influence the subsequent course of parasite growth in the animal.
The three immunized monkeys in Groups 1 and 2 whose pre-challenge antibody levels were above 150,000 (12504, 12518, and 12511) did not develop countable parasitemias whereas the other monkeys (12517, 12512, 12519) in these groups with pre-challenge antibody levels between 50,000 and 100,00 did develop countable - -parasitemias. These results suggest a strong relationship between the level of humoral immune response to SERA and the manifestation of immune protection.
The delayed appearance of a brief, low level, countable parasitemia in one monkey ~12512) in Group 2 was probably a consequence of changes in competing interactions of immunologic and physiologic factors at work in a partially immunized, infected monkey. The brevity and low level of the episode, which occurred during a long, fluctuating, low level parasitemia, does not suggest the appearance of a selected parasite mutant unresponsive to the anti-SERA immunity. The reason for the unexpectedly low parasitemia seen in the one animal of Group 3 is not known; it may reflect a natural resistance of some animals or a problem in injecting the parasite challenge.
The above results demonstrate that SERA 1 and y-IFN-SERA N polypeptides induce a protective immune response in ~g~y~ when immunization is done with CFA and - .

W092/0879~ 37 PCT/US90/0~61 2~3~55~

IFA. The y-IFN-SERA N provided a level of protection against infection (e.g., levels of parasitemia) and an immune response (e.g., antibody titers) that was similar to that of SERA 1.
3.h. De~osit of Bioloqical Materials Saccharomyces cerevisiae strain JSC302 host cells transformed with (a) pBS24Ub-SERA 1 and (b) pBS24Ub-y-IFN-SERA N have been deposited on November 7, 1990, with the American Type Culture Collection (ATCC), Rockville, MD, and designated as pBS24Ub-SERA 1 and pBS24Ub-y-IFN
SERA N and will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of patent procedure. Accession numbers are available from the ATCC.
These deposits are provided merely as convenience to those of skill in the art, and are not an admission that a deposit is required under 35 U.S.C. 112. The nucleic acid sequences of these plasmids, as well as the amino acid sequences of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with the description herein.
A license may be required to make, use, or sell the deposited materials, and no such license is hereby granted.

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Claims (32)

WHAT IS CLAIMED IS:

1. A nucleic acid construct comprising:
a first domain encoding ubiquitin; and a second domain encoding a malaria polypeptide or an epitope-containing fragment thereof, wherein the second domain is located downstream from the first domain.

2. The construct according to claim 1, wherein the nucleic acid is DNA.

3. The construct according to claim 2, wherein the malaria polypeptide is serine-repeat antigen (SERA) or an epitope-containing fragment thereof.

4. The construct according to claim 3, wherein the malaria polypeptide is an epitope-containing fragment of SERA.

5. The construct according to claim 4, wherein the malaria polypeptide is SERA 1.

6. The construct according to claim 1, further comprising a third domain encoding an immunomodulator polypeptide, wherein the third domain is located between the first and second domains.

7. The construct according to claim 6, wherein the immunomodulator polypeptide is human .gamma.-interferon.

8. The construct according to claim 7, wherein the malaria polypeptide is SERA or an epitope-containing fragment thereof.

9. The construct according to claim 8, wherein the malaria polypeptide is an epitope-containing fragment of SERA.

10. The construct according to claim 9, wherein the malaria polypeptide is SERA N.

11. A host cell transformed by the construct according to claim 1.

12. A yeast cell transformed by the construct according to claim 5.

13. A yeast cell transformed by the construct according to claim 10.

14. A fusion polypeptide comprising a first immunomodulator polypeptide and a second malaria polypeptide.

15. The fusion polypeptide according to claim 14, wherein the immunomodulator polypeptide is human .gamma.-interferon and the malaria polypeptide is SERA or an epitope-containing fragment thereof.

16. The fusion polypeptide according to claim 15, wherein the malaria polypeptide is SERA N.

17. An immunogenic composition comprising an immunogenic amount of SERA 1 admixed with a pharmaceutically acceptable vehicle.

18. The immunogenic composition according to claim 17, further comprising an adjuvant.

19. The immunogenic composition according to claim 18, further comprising an immunogenic amount of a non-SERA 1 malaria polypeptide.

20. The immunogenic composition according to claim 17, wherein the composition is a vaccine.

21. An immunogenic composition comprising an immunogenic amount of the fusion protein of claim 16 admixed with a pharmaceutically acceptable vehicle.

22. The immunogenic composition according to claim 21, further comprising an adjuvant.

23. The immunogenic composition according to claim 22, further comprising an immunogenic amount of a non-SERA N
malaria polypeptide.

24. The immunogenic composition according to claim 21, wherein the composition is a vaccine.

25. A method for prophylactic or therapeutic treatment of malaria comprising administering the vaccine of claim 20 to an individual.

26. A method for prophylactic or therapeutic treatment of malaria comprising administering the vaccine of claim 24 to an individual.

27. An immunoassay for detecting antibodies directed against a malaria antigen comprising:
(a) incubating a biological sample suspected of containing anti-malaria antibodies with a SERA 1 polypeptide, under conditions that allow the formation of an antibody-antigen complex; and (b) detecting the antibody-antigen complex.

28. An immunoassay for detecting antibodies directed against a malaria antigen comprising:
(a) incubating a biological sample suspected of containing anti-malaria antibodies with the fusion protein of claim 16, under conditions that allow the formation of an antibody-antigen complex; and (b) detecting the antibody-antigen complex.

29. A process for increasing the level of expression of a recombinant epitope-containing fragment of SERA in a yeast host cell comprising:
(a) constructing a vector consisting of a first domain encoding ubiquitin and a second domain encoding an epitope-containing SERA fragment, wherein the second domain is located downstream from the first domain;
(b) transforming a yeast host cell with the constructed vector of (a);
(c) culturing the transformed yeast host cell under conditions capable of inducing expression of the ubiquitin-SERA domains whereby the in vivo product is a recombinant SERA polypeptide; and (d) recovering the recombinant SERA polypeptide from the transformed yeast culture.

30. The process according to claim 29, wherein the SERA fragment is SERA 1.

31. A process for increasing the level of expression of a recombinant epitope-containing fragment of SERA in a yeast host cell comprising:

(a) constructing a vector consisting of a first domain encoding ubiquitin, a second domain downstream to the first domain encoding an epitope-containing SERA
fragment thereof, and a third domain located between the first and second domains encoding human .gamma.-interferon;
(b) transforming yeast with the constructed vector of (a);
(c) culturing the transformed yeast under conditions capable of inducing expression of the ubiquitin-human .gamma.-interferon-SERA domains, whereby the in vivo product is a recombinant human .gamma.-interferon-SERA
fusion polypeptide; and (d) recovering the recombinant fusion polypeptide from the transformed yeast culture.

32. The process according to claim 31, wherein the SERA fragment is SERA N.